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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 30, 2004 to December 28, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The discrepancy between originally usded identifier of the substance and new test material information is due to change in the main identifier through the Substance identity adaptation service offered by ECHA. The new identifier is covered by old identifier mentioned in Additional test material information section

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

-Source of inoculum/activated sludge: An aqueous phase inoculum of non adapted activated sludge was obtained from the municipal sewage treatment plant, D-31137 Hildesheim, which treats predominantly municipal sewage.
-Pretreatment: The activated sludge was filtered with folded filter. The first 200 mL of the filtrate was not used. The second filtrate effluent from the domestic waste water was used to initiate inoculation.
-Colony forming units in the test vessels: 104-106 CFU/L

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS:
- Composition of medium: The culture medium used in this study (mineral nutrient solution) was same as that recommended in the OECD Guidelines 301D
- Test temperature: 20-24 ± 1 °C
- pH: 7.4
- Continuous darkness: Yes, in an incubator

TEST SYSTEM:

- Culturing apparatus: 300 mL BOD bottles with glass stoppers
- Number of culture flasks: 5 x 2 each for test substance, reference substance and inoculum control and for toxicity control
- Test performed in closed vessels: Yes
- Method used to create aerobic conditions: 1 day before the test demineralised water for the test medium was aerated until oxygen saturation and thereafter left at room temperature for 20 hours.
- Other: All test solutions were made as stocks solutions.The referance substance was weighed directly. Test substance was prepared as a stock solution. 0.2 mL inoculum was added to each BOD bottle.

SAMPLING:
- Sampling frequency: Samples were taken from the BOD bottles on Days 0, 7, 14, 21 and 28 days for O2 determination, BOD determination and percentage degradation.

-Other: On Days 0 and 28, the O2 concentration and the pH-values of all the inoculum control, functional control, tetst substance and toxicity control was carried out.
- The temperature inside the incubator was checked every working day.

ANALYSIS:
- Measuring equipments: pH-meter, corning pH 240, Oximeter, WTW Oxi 530, Incubator, Rubarth.

CONTROL AND BLANK SYSTEM:
- Inoculum blank: Yes ( test flasks containing inoculum and nutrient solution)
- Toxicity control: Yes; (test flask containing 45 mL test substance solution/ 3L + 5 mg/L reference item + inoculum + nutrient solution)
- Reference control: Yes; (test flasks containing 10 mg/L of reference substance, i.e., sodium acetate)

Reference substance:

acetic acid, sodium salt

Remarks:

at 10 mg/L (ThOD = 0.78 mg02/mg)

Preliminary study:

None

Test performance:

No unusual obervations were made during the test.

Key result

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

28 d

Details on results:

- The oxygen demand of the inoculum control was 0.97 mgO2/L after 28 days
- For the functional control adaptation phase transformed to a degradation phase with >10% degradation after 1 day, >60% after 5 days and 96% after 28 days. Hence clearing the validity criteria.
- The biodegradation of the toxicity control was 51% after 14 days and 81% after after 28 days. Hence, the biodegradation of the reference substance was not inhibited by the test substance.
- The test substance reached 10% degradation after 2 days, pass level of >60% after 26 days and a maximum degradation of 70% after 28 days

Results with reference substance:

Approximately 96 % degradation was observed after 28 d:

Refer attached background material for details.

Validity criteria fulfilment:

- The percentage degradation of the functional control reached the pass level of >60% after 14 d.

- The oxygen depletion in the inoculum control came to 0.97 mg dissolved oxygen/L after 28 d.

- The residual concentration of oxygen in the test bottles did not fall below 0.5 mg/L at any time.

- The difference of extremes of replicate values of removal of the test substance at the end of the test was less than 20 %.

- The percentage degradation of the toxicity control reached the pass level of 25 % after 14 d.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the test substance was considered to be readily biodegradable.

Executive summary:

A study was performed to assess the ready biodegradability of the test substance according to OECD Guideline 301 D, in compliance with GLP. Activated sewage sludge was exposed to the substance at a concentration of 3.0 mg/L in BOD bottles in the dark at 20–24°C for 28 d. Degradation was assessed by determination of oxygen consumption, expressed as percentage BOD. Biodegradation attained 70% after 28 d. The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time. All other validity criteria were fulfilled. Under the conditions of the study, the test substance was considered to be readily biodegradable (Noack, 2005).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From December 20, 2005 to January 17, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The discrepancy between originally usded identifier of the substance and new test material information is due to change in the main identifier through the Substance identity adaptation service offered by ECHA. The new identifier is covered by old identifier mentioned in Additional test material information section

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Organisms from an activated laboratory sewage treatment were used for inoculum. Fresh samples from a lake, the effluent of a municipal sewage treatment plant and an extract of surface soil were mixed and cultivated in a laboratory sewage treatment.
- Method of cultivation: A composed inoculum was used and air germs was added via aeration. The sludge unit was operated to good practice: effluents were clear and the temperature was kept between 20 and 25 °C. The sludge settled well and aeration kept the mixture aerobic at all times.
- Storage length: The sludge was used as inoculum until after at least one month's operation, but not after more than four months. Thereafter samples from the above mentioned sites were collected again, mixed with an aliquot of the old sludge and cultivated as described above.
- Preparation of inoculum for exposure: A fresh sample from the Laboratory model activated sludge plant containing approximately 1.875 g suspended solids was collected and filtered by means of a water jet pump through a filter paper. The sludge was washed twice with mineral medium. Afterwards it was suspended in 250 mL mineral medium and stirred until it was homogeneous. 4 mL of this solution were used to inoculate 1 L of mineral medium. This amount of inoculums corresponded to 30 mg/L suspended solids.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineralised medium 10 mL of solution A and 1 mL each of of solution B, C and D are mixed with 800 mL of the demineralised water and volume was made up to 1000 mL (for details see attached background material).
- Suspended solids concentration: 30 mg/L
- pH: 7.4 ± 0.2

TEST SYSTEM
- Culturing apparatus: Closed respirometer flask
- Number of culture flasks/concentration: Three for inoculum blank and test substance; two each for abiotic control, reference control and toxicity control.
- Measuring equipment: Sensomat system (Digital Respirometer System, Liebherr, Aqualytic, Langen, Germany) at 22 ± 1°C.
- Details of trap for CO2 and volatile organics if used: CO2 produced was trapped in potassium hydroxide.

SAMPLING
- Sampling frequency: Every day

CONTROL AND BLANK SYSTEM (for details refer to attached background material)
- Inoculum blank: Containing only inoculum but without test substance
- Abiotic degradation control: Yes, a solution with a concentration of 100 mg/L test substance in mineral medium.
- Reference control: Yes, 100 mg/L sodium benzoate in inoculated mineral medium.
-Toxicity control: Yes, a solution of 100 mg/L test substance and 100 mg/L sodium benzoate in inoculated mineral medium.

-Other:
- N-allylthiourea (N-ATH) was added (in amounts depending on the COD of the test or referance susbtance) into the test vessel to avoid nitrification.
- Mercury (II) chloride (as solution) was added to the abiotic controls in a concentration of 0.01 g/L.
- pH values of all the test vessels: Refer to attached backgroud materail for details

Reference substance:

benzoic acid, sodium salt

Remarks:

(tested at 100 mg/L concentration)

Preliminary study:

Determination of the Chemical Oxygen Demand (COD) of the test and reference substance in the separate studies and was found to be 2,258 mg O2/g and 1,638 mg O2/g, respectively.

Test performance:

No unusual observations are made during the course of the study.

Key result

Parameter:

% degradation (O2 consumption)

Value:

58

St. dev.:

1

Sampling time:

28 d

Details on results:

Results:
- There were no deviations in the environmental test conditions with regard to the temperature which was within the pre-set range of 22 ± 1 °C.
- The functional capability of the inoculum was sufficient, as the reference substance sodium benzoate was biodegraded to an average of 83 % within 14 days.
- Inoculum blanks reached a mean oxygen consumption of 27.7 mg per litre within 28 days.
- The test substance is not assumed to be inhibitory, because 40 % degradation (based on total ThoD or COD, respectively) occurred within 14 days in the toxi
city test.
- The test parameters indicate the validity of the experiment, because the requirements were fulfilled. Unusual observations were not made.
- The average biodegradation value of test substance was 58 % after 28 days. However, the test substance was biodegraded to only 34 % in a 10d window within the 28 days period. There was no relevant adaptation time for the polyvalent microorganisms necessary to start the biodegradation process. Hence, the test substance cannot be regarded as readily biodegradable. Nevertheless, it can can be regarded as moderately biodegradable.

Results with reference substance:

Approximately 83% degradation was observed after 28 d:

The functional capability of the inoculum was sufficient, as the reference substance sodium benzoate was biodegraded to an average of 83% within 14 days. Inoculum blanks reached a mean oxygen consumption of 27.7 mg /L within 28 days. The test substance is not assumed to be inhibitory, because 40 % degradation occurred within 14 days in the toxicity test.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Under the conditions of the study, the test substance was not considered to be readily biodegradable but could be regarded as moderately biodegradable.

Executive summary:

A study was conducted to evaluate the ready biodegradability of the test substance according to OECD Guideline 301 F and EU Method C.4-D, in compliance with GLP. Activated sludge was inoculated with the test (100 mg/L) or reference (benzoic acid, sodium salt; 100 mg/L) substance, kept in darkened, enclosed respirometer flasks (22 ± 1°C) and observed for degradation by measurement of the dissolved oxygen concentration over a 28 d period. Biodegradation of the test and reference substance was determined to be 58 and 83%, respectively, after 28 d. Therefore, under the conditions of the study, the test substance was not considered to be readily biodegradable but could be regarded as moderately biodegradable (Wehrhahn, 2006).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 April 2003 to 7 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(according to OECD, EC German principles of GLP)

Specific details on test material used for the study:

The discrepancy between originally usded identifier of the substance and new test material information is due to change in the main identifier through the Substance identity adaptation service offered by ECHA. The new identifier is covered by old identifier mentioned in Additional test material information section

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from municipal sewage treatment plant, D-31137 Hildesheim
- Preparation of inoculum for exposure: The activated sludge was maintained in an aerobic condition by aeration for 4 h and then homogenized with a mixer. The sludge was filtered and the filtrate (30 mL) was subsequently used to initiate inoculation.
- Initial cell/biomass concentration: 10E7 - 10E8 CFU/mL

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

act. ingr.

Initial conc.:

67.9 other: %

Based on:

other: TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Test temperature: 20-24°C
- pH: The pH of media at the beginning of the test is not reported. However, the pH-value of all solutions was measured on the last day of the test (Day 28). The pH values for inoculum control, functional control and test flask at 15 mg/L were 7.14 (mean of 2 replicates), 7.44 and 7.13 (mean of 2 replicates) respectively.
- Aeration of dilution water: Before the start of the test, the mineral nutrient solution with inoculum was aerated for 24 h with CO2-free air to purge the system of CO2.
- Composition of medium: Mineral nutrient solution according to OECD 301 B/CO2 Evolution test

TEST SYSTEM
- Culturing apparatus: Brown glass bottles (5000 mL) as incubation vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The vessels were connected to the system for the production of CO2-free air and aerated for 24 h.
- Measuring equipment: The room temperature was measured continuously by a hygrothermograph. CO2 determination was carried out by titration subsequent to complete adsorption of the released CO2 in a basic solution. Back titration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly. On the 28th day the pH of all solutions were measured prior to acidification.
- Details of trap for CO2 and volatile organics if used: The CO2-adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles.

SAMPLING
- Sampling frequency: On Day 1, 3, 6, 7, 9, 14, 17, 21, 24, 28 and 29
- Other: On the 28th day 1 mL concentrated HCl was added to each of the vessels. Aeration was continued for a further 24 h and on the 29th day the quantity of CO2 released in the last two gas wash bottles was determined.

CONTROL AND BLANK SYSTEM
- Inoculum control: Yes
- Toxicity control: Yes

Reference substance:

acetic acid, sodium salt

Remarks:

Batch # 420773/153701, CAS # 127-09-3, Purity >99%, Replicate: Single, Test concentration: 35 mg/L, ThCO2: 1.07 mg CO2/mg, ThTOC: 0.29 mg C/mg, Carbon content in the vessel: 10.2 mg C/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

86

Sampling time:

28 d

Remarks on result:

other: Mean of 2 replicates

Details on results:

Carbon content of the test substance: Based on the carbon content a ThCO2 of 2.49 mg CO2/mg test substance was calculated. On this basis the test concentration of 15 mg/L, corresponding to a carbon content of 10.2 mgC/L in the test vessels was selected.

CO2-production and biodegradation: The total amount of CO2 produced in 28 d was analysed by titration in 12 measurements.
- In the control group a maximum of 43.2 mg CO2/L was formed after 28 d (validity criterion: <70 mg CO2/L after 28 d.
- In the test group gross CO2 production after 28 d was 69.9 mg/L (replicate 1) and 89.4 mg/L (replicate 2). The 10% biodegradation level was reached after 5 days. In the 10-d-window the pass level of a biodegradation of more than 60% was reached. The mean biodegradation came to 79% after 14 d and to a maximum of 86% after 28 d.
- In the toxicity control 62% biodegradation occurred within 14 d and came to a maximum of 78% after 28 d. The test substance did not inhibit the biodegradation of the reference substance.

Results with reference substance:

The adaptation phase of the functional control group changed after 2 d into the degradation phase (degradation ≥ 10%). The course of the degradation phase was rapid and reached a degradation rate of 60% on Day 7. The validity criterion of a degradation of 60% after 14 d was fulfilled.

Criteria met for the validity of the study:

- The total CO2 evolution in the inoculum blank at the end of the test did not exceed 70 mg/L.

- The biodegradation of the reference compound reached the pass level of ≥60 % by Day 14.

- The difference of extremes of replicate values of the test substance at the end of the 10-d window was less than 20%.

Table 1: Biodegradation of the test substance

	Biodegradation [%]
	Study day
	6	14	21	28
Test substance (15 mg/L)	37	79	86	86
Reference substance (35 mg/L)	56	100	100	100
Toxicity control (15 mg/L test substance + 35 mg/L reference substance)	31	62	67	78

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of the study, the test substance was considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance in the CO2-evolution test according to OECD Guideline 301 B (modified Sturm test), in compliance with GLP. A solution of the test substance (15 mg/L) in nutrient solution, corresponding to 10.2 mg TOC/L, was inoculated with activated sludge. The test vessels were aerated by the passage of CO2-free air and incubated under aerobic conditions for 28 d. The biodegradation of the test substance was followed by titrimetric analyses of the quantity of CO2 produced on Days 1, 3, 6, 7, 9, 14, 17, 21, 24, 28 and 29 of the study. The reference substance used was sodium acetate at a concentration of 35 mg/L. The reference substance reached the pass levels for ready biodegradability after 7 d. The mean biodegradation of the test substance was 79% after 14 d and reached a maximum of 86% after 28 d (after acidification). Under the conditions of the study, the test substance was considered readily biodegradable (Noack, 2003).

Reference 4

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 5 November 2001 to 3 December 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

The discrepancy between originally usded identifier of the substance and new test material information is due to change in the main identifier through the Substance identity adaptation service offered by ECHA. The new identifier is covered by old identifier mentioned in Additional test material information section

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from sewage treatment plant Frankfurt-Niederrad
- Pretreatment: The activated sludge was aerated for 1 d and washed three times with mineral medium.
- Concentration of sludge: 8 g centrifuged activated sludge/L

Duration of test (contact time):

28 d

Initial conc.:

152.5 mg/L

Based on:

act. ingr.

Initial conc.:

94.7 mg/L

Based on:

other: DOC

Initial conc.:

151.1 mg/L

Based on:

act. ingr.

Initial conc.:

93.8 mg/L

Based on:

other: DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 302B.
- Test temperature: 22 ± 2°C
- Other: Incubation took place in diffuse light.

TEST SYSTEM
- Culturing apparatus: Beaker (Capacity 4L)
- Test volume: 2L
- Number of culture flasks/concentration: One vessel for each concentration of the test substance
- Other: A toxicity test, containing both the test substance and the reference substance, was not run because the test substance was assumed not to be inhibitory.

SAMPLING
- Sampling frequency: On Days 0, 0.125 (3 h), 3, 7, 14, 21 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

Reference substance:

diethylene glycol

Remarks:

(Purity: >99% (m/m), Number of determinations: Single, Test concentration: 200.7 mg/L, ThDOC: 0.45 mg C/mg substance, ThDOC in the test vessel: 90.3 mg C/L)

Key result

Parameter:

% degradation (DOC removal)

Value:

95

Sampling time:

28 d

Remarks on result:

other: test substance concentration at 152.4 mg/L

Key result

Parameter:

% degradation (DOC removal)

Value:

97

Sampling time:

28 d

Remarks on result:

other: test substance concentration at 151.1 mg/L

Details on results:

The measured DOC concentrations of test solutions after 28 d were: 7.9 and 7.2 mg/L for 152.5 and 151.1 mg/L respectively.

Results with reference substance:

- The reference substance met the validity criteria for the test, greater than 70% biodegradation within 14 d (Mean % degradation on Day 3 = 0%, Day 7 = 92%, Day 14 = 98% and Day 29 = 99%).
- The measured DOC concentration of reference substance after 28 d was found to be 6.4 mg/L.

Examination of the validity criteria:

The validity criteria were not completely fulfilled according to the guideline:

- The reference substance reached the pass level of 70% degradation within 14 d.

- The test substance showed gradual DOC removal over the test period.

- The DOC elimination of the test substance after 3 h should be less than 20%. However, the DOC elimination after 3 h was 25%.

Table 1: DOC content and biodegradation of the test substance

Study day [d]	DOC [mg/L]		Biodegradation [%]
	S1	S2	S1	S2
0	82.8	81.6	-	-
0.125	67.4	74.8	-	-
3	21.4	19.3	73	79
7	12.7	10.5	88	92
14	6.4	6.6	97	97
21	6.4	6.5	97	97
28	7.9	7.2	95	97

 

S1 = Test substance concentration at 152.5 mg/L

S2= Test substance concentration at 151.1 mg/L test substance

Validity criteria fulfilled:

no

Remarks:

(with the exception that the DOC elimination after 3 h was 25% instead of <20%)

Interpretation of results:

inherently biodegradable

Conclusions:

Under the conditions of the study, the substance was considered to be inherenty biodegradable.

Executive summary:

A study was conducted to determine the biodegradability of the test substance according to OECD Guideline 302 B (Zahn Wellens/EMPA test). The test substance reached a biodegradation of 96% after 28 d. The reference substance reached the pass levels for ready biodegradability after 14 d. The validity criteria according to the guideline were not completely fulfilled in the study. The elimination of the test substance after 3 h was relatively high (25%) and initial DOC content was significantly lower than expected. Therefore it was concluded physicochemical adsorption played a role in the elimination of the test substance. Under the conditions of the study, the substance was considered to be inherently biodegradable (Hirschen and Grohmann, 2002).

Description of key information

Overall, based on all available data, the test substance is considered as readily biodegradable. 

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

A study was conducted to determine the ready biodegradability of the test substance in the CO2-evolution test according to OECD Guideline 301 B (modified Sturm test), in compliance with GLP. A solution of the test substance (15 mg/L) in nutrient solution, corresponding to 10.2 mg TOC/L, was inoculated with activated sludge. The test vessels were aerated by the passage of CO2-free air and incubated under aerobic conditions for 28 d. The biodegradation of the test substance was followed by titrimetric analyses of the quantity of CO2 produced on Days 1, 3, 6, 7, 9, 14, 17, 21, 24, 28 and 29 of the study. The reference substance used was sodium acetate at a concentration of 35 mg/L. The reference substance reached the pass levels for ready biodegradability after 7 d. The mean biodegradation of the test substance was 79% after 14 d and reached a maximum of 86% after 28 d (after acidification). Under the conditions of the study, the test substance was considered readily biodegradable (Noack, 2003).

A study was performed to assess the ready biodegradability of the test substance according to OECD Guideline 301 D, in compliance with GLP. Activated sewage sludge was exposed to the substance at a concentration of 3.0 mg/L in BOD bottles in the dark at 20–24°C for 28 d. Degradation was assessed by determination of oxygen consumption, expressed as percentage BOD. Biodegradation attained 70% after 28 d. The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time. All other validity criteria were fulfilled. Under the conditions of the study, the test substance was considered to be readily biodegradable (Noack, 2005).

A study was conducted to evaluate the ready biodegradability of the test substance according to OECD Guideline 301 F and EU Method C.4-D, in compliance with GLP. Activated sludge was inoculated with the test (100 mg/L) or reference (benzoic acid, sodium salt; 100 mg/L) substance, kept in darkened, enclosed respirometer flasks (22 ± 1°C) and observed for degradation by measurement of the dissolved oxygen concentration over a 28 d period. Biodegradation of the test and reference substance was determined to be 58 and 83%, respectively, after 28 d. Therefore, under the conditions of the study, the test substance was not considered to be readily biodegradable but could be regarded as moderately biodegradable (Wehrhahn, 2006).

A study was conducted to determine the biodegradability of the test substance according to OECD Guideline 302 B (Zahn Wellens/EMPA test). The test substance reached a biodegradation of 96% after 28 d. The reference substance reached the pass levels for ready biodegradability after 14 d. The validity criteria according to the guideline were not completely fulfilled in the study. The elimination of the test substance after 3 h was relatively high (25%) and initial DOC content was significantly lower than expected. Therefore it was concluded that physicochemical adsorption played a role in the elimination of the test substance. Under the conditions of the study, the substance was considered to be inherently biodegradable (Hirschen and Grohmann, 2002).